Abstract The heterotrimeric G protein-mediated signal transduction pathways play an important regulatory role in the human pathogenic fungus Cryptococcus neoformans. The Ga subunit Gpa1 governs a conserved cAMPdependent signaling pathway that governs virulence, but the necessary components of the Gpa1 heterotrimeric G protein complex are not known, suggesting that Gpa1 signaling may be atypical. Using Gpa1 as bait and through a yeast two-hybrid screen, we have identified a novel G[unreadable]-like/RACK1 protein homolog, Gib2, which exhibits a G[unreadable]-like function by association with Gpa1 and G? subunits Gpg1 and Gpg2. In addition, we found that Gib2 regulates the intracellular level of cAMP in a gpa1 mutant strain that is defective in cAMP signaling and that Gib2 is required for full virulence expression in a murine model of encephalitis. Moreover, we found that Gib2 interacts with additional proteins of importance, such as the novel homolog of the human intersectin 1 protein Cin1 that regulates morphogenesis and growth of C. neoformans. Four specific aims are proposed to illustrate the mechanisms by which Gib2 interacts with Gpa1 and Gpg1/Gpg2, regulates intracellular cAMP levels and virulence, and functions as a multi-faceted protein by interacting with Cin1. Identification and characterization of Gib2-mediated atypical G protein signaling and Gib2 functions are significant not only for our understanding of signal transduction mechanisms regulating cellular growth, differentiation, and pathogenesis, but also in our pursuit of novel targets for antifungal therapy.